1. Field of the Invention
The present invention relates to medical diagnostics for tissue investigation and more particularly, it relates instruments for real-time monitoring of tissues in vivo.
2. Description of Related Art
Esophageal adenocarcinoma will claim approximately 89.6% of all cases within 5 years of diagnosis [2,3]. This rare but fatal disease has been increasing in the United States at the alarming rate of approximately 2% every year, and has been linked to conditions such as obesity [3-7]. A significant factor of the poor prognosis is the difficulty of early detection when disease initiates in the epithelial tissues before becoming invasive. The epithelial layer may contain intestinal metaplasia (Barrett's esophagus), low-grade dysplasia, high-grade dysplasia, and/or carcinoma in heterogeneous (initiation) sites. Additionally, onset is often asymptomatic until the disease has advanced to the point where the curative therapy may be unavailable. This asymptomatic, and potentially heterogeneous nature of diseased esophageal mucosa makes early detection of abnormal progression almost impossible to distinguish using current endoscopic surveillance of the esophagus [8]. Esophageal surveillance is presently done using standard white light video endoscopy (SVE) and/or chromoendoscopy, the use of contrast agents such as acetic acid or methylene blue [9]. Current SVE procedures rely on visual assessment of the tissue mucosa followed by random 4 quadrant biopsy of suspicious epithelial tissue [8-11]. Often times, dysplastic progression is invisible to SVE due to low resolution, and missed during random sampling. This random sampling is of great concern due to the inherent sampling error and delayed turn-around time for results [12]. The excised tissue is preserved and prepared for histologic evaluation of microstructure change; however, discrepancies in histopathology readings prompted a modification of standard esophageal classification [13]. Presently, a large amount of research is being exerted in the optical field to develop early detection screening systems for these premalignant conditions.
Fluorescence optical spectroscopy under ultra violet (UV) light has been shown to provide a powerful method for minimally invasive detection of biochemical and morphological changes [14-16] in regions of the gastrointestinal (GI) tract [11,17,18]. While spectroscopy is very sensitive in detecting these changes, the point measurements are averaged over an area and do not provide a comprehensive image. Diagnostic information would be better relayed and more familiar to the surgical team as an image. Time resolved imaging techniques such as time correlated single photon counting (TCSPC) and fluorescence lifetime imaging (FLIM)) have been explored [19]. Investigators acknowledged the difficulties in fitting multiple exponentials to resulting data, as well as achieving the required resolution with these techniques.
Optical coherence tomography (OCT) provides images of major structural components of the mucosa and submucosa, including esophageal glands, intestinal villi, colonic crypts and blood vessels, at higher resolution than catheter probe endoscopic ultrasound (CPEUS) [20]. While these features allow visibility of normal squamous mucosa and specialized intestinal metaplasia in the esophagus, OCT has not yet been shown to adequately differentiate between dysplastic and intramucosal carcinoma [21].
Magnification high-resolution endoscopy is commercially available from Olympus [22-24]. This wide-field zoom endoscope system provides images of mucosal and vascular patterns by implementing white light (WL), autofluorescence (AF), and/or narrowband imaging (NBI). Results suggest an increase in lesion detection, although it was not shown to provide cellular imaging.
Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. This technique has gained popularity in the scientific and industrial communities and typical applications are in life sciences and semiconductor inspection. The principle of confocal imaging was patented by Marvin Minsky in 1957 and aims to overcome some limitations of traditional wide-field fluorescence microscopes. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded in light from a light source. In the case of imaging a tissue specimen, the excitation light has an average photon penetration depth that is strongly dependant on wavelength and it is on the order of about 2 mm at 400 nm excitation to more than 1 cm in the near infrared spectrum. The resulting fluorescence arises from all regions of the specimen that are illuminated by the excitation light and subsequently, the depth of the tissue region that produces fluorescence is about equal to the average photon penetration depth of the excitation light. This fluorescence is detected by the microscope's photodetector or camera. However, as the image plane of a conventional microscope has a thickness on the order of 10-50 μm, nearly all the signal collected by the microscope's optics is out of focus, thus not contributing to the formation of an image but act as background signal and is equivalent to a background noise. This problem is further enhanced by multiple scattering of the photons arriving from deeper tissue layers resulting in a loss of the image information. In contrast, the confocal microscope eliminated this “background signal” by using point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information—the name “confocal” stems from this configuration. As only fluorescence very close to the focal plane can be detected, the image contrast, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, due to the fact that much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity, and is more time intensive as it requires image formation via point-by-point scanning. The long exposure time effectively eliminates most, in not all, in vivo uses when tissue autofluorescence is used as the primary signal for image formation, due to the fact that living tissue is always moving to some degree. In addition, the ability of an operator to keep a microscope system to a still (no motion) position is also limited and practically impossible when microscopic resolution is needed. Furthermore, for in-vivo application, the scanning speed for image acquisition directly determines how large an area within the region of interest (such as esophagus) can be examined with the microscope because the time available in the OR is limited and very expensive. Pentax Corporation and Mauna Kea Technologies both commercially distribute a confocal endomicroscope system [25,26]. While in vivo cellular imaging was achieved, the use of fluorescein was required for both. The results are thus dependent on fluorescein uptake, concentration, leakage, pattern distortion, and other complications. Additionally, it was noted that encasement of the individual white light and confocal optics at the distal end of the endoscope limited the ability of the white light image to guide placement of the confocal collection point [26].
Confocal microscopy has been utilized to characterize tissue autofluorescence of frozen esophageal biopsies [27]. While histopathologically comparable images were obtained from the sectioned and stained samples, the destructive nature in preparing the sample eliminates this approach from in vivo application. Confocal techniques have also been implemented in a dual axes configuration to image fresh tissue [28,29]. The first prototype used acetic acid and was shown to provide ex vivo images of glandular crypt size and organization, although nuclei were not specified to be distinguishable. The second prototype used fluorescein to obtain in vivo images of blood vessels, but suffered from leakage over time and did not provide cellular images. The advancement toward endoscopic implementation of microscopy is understood to be a key towards endoluminal visualization.
Endoscopic confocal [30], or endoscopic wide-field [31] microscopy implement fluorescein and acriflavine hydrochloride contrast agents respectively. While visualization of the nuclei is critical for diagnosis, contrast enhancement using exogenous agents raise concerns about allergic reactions, non-specific binding, saturation [8], dosage and toxicity levels, or delay due to chemical synthesis, manufacture, or processing [32].
Backscattered second harmonic generation and two-photon AF microscopy have been used to image the esophagus using near infrared (NIR) wavelengths without the use of contrast agents [33]. In addition to requiring complex nonlinear instrumentation, these methods focus on the esophageal stroma rather than the nuclei.
Accordingly, endoscopic methods for implementation of AF (i.e., without exogenous agents) in vivo microscopy to effectively visualize and categorize various abnormal esophageal tissue forms is understood to be a key towards endoluminal visualization.